NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification

Comparison and transfer testing of multiplex ligation detection methods for GM plants

<p>Abstract</p> <p>Background</p> <p>With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.</p> <p>Results</p> <p>Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).</p> <p>Conclusions</p> <p>From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.</p

Etude des relations structure/fonction d'une bactériocine anti-Listeria, la mésentéricine Y105

Mesentericin Y105 is a sub-class IIa bacteriocin produced by Leuconostoc mesenteroides Y105. In an attempt to understand the mecanism of its anti-listerial activity, we investigated the peptides structure-function relationship. For this purpose, a bank of mesentericins Y105 peptide derivates was created with modifications at single positions of the parent peptide. Peptide derivatives were generated using, randomly primed PCR mutagenesis to create single mutations in the DNA sequences that encode the mature peptide. A method for heterologous production based on a two componant system with one vector harboring structural gene, and one other encoding the immunity protein and transport system was optimized .Thus, an universal peptide secretion tool has been established. The impact of mutations was studied for the mesentericin Y105 derivatives, by accessing there activity, secondary structure (obtained by circular dichroism in trifluoroethanol and lysophosphatidylcholin micelles), predicted tertiary structure and interaction with anisotropic environment (method of tryptophan blue shift). Finally, a nuclear magnetic resonance analysis was used to determinate the native bacteriocin structure. A general model for the mechanism of action of mesentericin Y105 and its related bacteriocins is proposed.La mésentéricine Y105 est une bactériocine de sous-classe IIa produite par Leuconostoc mesenteroides Y105. Pour comprendre le mécanisme de l'activité anti-Listeria de ce peptide et plus généralement des bactériocines de classe IIa, l'étude des relations existant entre la structure et la fonction de ce peptide a été envisagée. Dans ce but, une collection de mésentéricines Y105 modifiées au niveau d'un résidu a été produite. Afin d'obtenir ces dérivés de bactériocine, une méthode de mutagenèse aléatoire par PCR a été mise en place pour générer des séquences d'ADN, codant la bactériocine mature, modifiées sur un seul codon. Dans un deuxième temps, une méthode de production hétérologue de ces peptides mutés a été développée en utilisant d'une part un vecteur portant les gènes de structure des bactériocines, et d'autre part, un vecteur permettant l'expression de l'immunité et du transport de ces peptides. Un outil de production universelle de peptide a été élaboré. L'étude de l'impact des mutations sur l'activité antagoniste, la structure secondaire (analysée par dichroïsme circulaire en présence de trifluoroéthanol ou de micelles de lysophosphatidylcholine), la structure tridimensionnelle (prédite) et l'interaction de la mésentéricine Y105 avec des environnements mimant les membranes cibles (méthode d'extinction de la fluorescence intrinsèque du tryptophane) a été réalisée. Enfin, une analyse par résonance magnétique nucléaire (RMN) a été effectuée sur la bactériocine sauvage pour déterminer sa structure tridimensionnelle. De l'ensemble de ces données, un modèle d'action est proposé pour la mésentéricine Y105, ce modèle peut être étendu aux bactériocines de structure proche

Etude des relations structure / fonction d'une bactériocine anti-listeria, la mésentéricine Y105

La mésentéricine Y105 est une bactériocine de sous-classe IIa produite par Leuconostoc mesenteroides Y105. Pour comprendre le mécanisme de l'activité anti-Listeria de ce peptide, l'étude structure-fonction de ce peptide a été envisagée. Dans ce but, une collection de mésentéricines Y105 modifiées a été produite. Afin d'obtenir ces dérivés, une méthode de mutagenèse aléatoire par PCR a été mise en place. Puis, une méthode de production hétérologue de ces peptides mutés par un système à deux plasmides a été développée. L'étude de l'impact des mutations sur l'activité antagoniste, la structure secondaire (analysée par dichroïsme), la structure tridimensionnelle et l'interaction de la mésentéricine Y105 avec des environnements mimant les membranes cibles (fluorescence intrinsèque du tryptophane) a été réalisée. Enfin, une analyse par résonance magnétique nucléaire a été effectuée sur la bactériocine native pour déterminer sa structure tridimensionnelle. De l'ensemble de ces données, un modèle d'action est proposé pour la mésentéricine Y105, ce modèle peut être étendu aux bactériocines de structure proche.Mesentericin Y105 is a sub-class IIa bacteriocin produced by Leuconostoc mesenteroides Y105. In an attempt to understand the mecanism of its anti-listerial activity, we investigated the peptide structure-function relationship. For this purpose, a bank of mesentericins Y105 peptide derivates was created. Peptide derivatives were generated using, randomly primed PCR mutagenesis. A method for heterologous production based on a two componant system was optimized. The impact of mutations was studied for the mesentericin Y105 derivatives, by accessing there; activity, secondary structure (obtained by circular dichroism), predicted tertiary structure and interaction with anisotropic environment (tryptophan blue shift). Finally, a nuclear magnetic resonance analysis was used to determinate the native bacteriocin structure. A general model for the mechanism of action of mesentericin Y105 and its related bacteriocins is proposed.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Human Pre-Elafin Inhibits a Pseudomonas aeruginosa-Secreted Peptidase and Prevents Its Proliferation in Complex Media▿

Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gram-negative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated